Actively helping customers, employees and the global community during the coronavirus SARS-CoV-2 outbreak.  Learn more >>

iFluor® 647 maleimide

HeLa cells were stained with mouse anti-tubulin followed with iFluor<sup>TM</sup> 647 goat anti-mouse IgG (H+L) (red); and nuclei were stained with DAPI (blue).
HeLa cells were stained with mouse anti-tubulin followed with iFluor<sup>TM</sup> 647 goat anti-mouse IgG (H+L) (red); and nuclei were stained with DAPI (blue).
Ordering information
Price ()
Catalog Number1065
Unit Size
Find Distributor
Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
Molecular weight1211.35
SolventDMSO
Spectral properties
Correction Factor (260 nm)0.03
Correction Factor (280 nm)0.03
Correction Factor (656 nm)0.0793
Extinction coefficient (cm -1 M -1)2500001
Excitation (nm)656
Emission (nm)670
Quantum yield0.251
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12171501

OverviewpdfSDSpdfProtocol


Molecular weight
1211.35
Correction Factor (260 nm)
0.03
Correction Factor (280 nm)
0.03
Correction Factor (656 nm)
0.0793
Extinction coefficient (cm -1 M -1)
2500001
Excitation (nm)
656
Emission (nm)
670
Quantum yield
0.251
AAT Bioquest's iFluor® dyes are optimized for labeling proteins, in particular, antibodies. These dyes are bright, photostable and have minimal quenching on proteins. They can be well excited by the major laser lines of fluorescence instruments (e.g., 350, 405, 488, 555 and 633 nm). iFluor® 555 dyes have fluorescence excitation and emission maxima of ~550 nm and ~570 nm respectively. iFluor® 647 family has the spectral properties essentially identical to those of Cy5® (Cy5® is the trademark of GE Healthcare). Compared to Cy5 probes iFluor® 647 family has much stronger fluorescence and higher photostability. Their fluorescence is pH-independent from pH 3 to 11. These spectral characteristics make this new dye family an excellent alternative to Cy5® and Alexa Fluor® 647 (Cy5® and Alexa Fluor® are the trademark of Invitrogen and GE Health Care) . iFluor® 647 maleimide is reasonably stable and shows good reactivity and selectivity with thiol group.

Example protocol


PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. iFluor™ 647 maleimide stock solution (Solution B)
Add anhydrous DMSO into the vial of iFluor™ 647 maleimide to make a 10 mM stock solution. Mix well by pipetting or vortex.
Note     Prepare the dye stock solution (Solution B) before starting the conjugation. Use promptly. Extended storage of the dye stock solution may reduce the dye activity. Solution B can be stored in freezer for upto 4 weeks when kept from light and moisture. Avoid freeze-thaw cycles.


2. Protein stock solution (Solution A)
Mix 100 µL of a reaction buffer (e.g., 100 mM MES buffer with pH ~6.0) with 900 µL of the target protein solution (e.g. antibody, protein concentration >2 mg/mL if possible) to give 1 mL protein labeling stock solution.
Note     The pH of the protein solution (Solution A) should be 6.5 ± 0.5.
Note     Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or other proteins will not be labeled well.
Note     The conjugation efficiency is significantly reduced if the protein concentration is less than 2 mg/mL. For optimal labeling efficiency the final protein concentration range of 2-10 mg/mL is recommended.

Optional: if your protein does not contain a free cysteine, you must treat your protein with DTT or TCEP to generate a thiol group. DTT or TCEP are used for converting a disulfide bond to two free thiol groups. If DTT is used you must remove free DTT by dialysis or gel filtration before conjugating a dye maleimide to your protein. Following is a sample protocol for generating a free thiol group:
  1. Prepare a fresh solution of 1 M DTT (15.4 mg/100 µL) in distilled water.
  2. Make IgG solution in 20 mM DTT: add 20 µL of DTT stock per ml of IgG solution while mixing. Let stand at room temp for 30 minutes without additional mixing (to minimize reoxidation of cysteines to cystines).
  3. Pass the reduced IgG over a filtration column pre-equilibrated with "Exchange Buffer". Collect 0.25 mL fractions off the column.
  4. Determine the protein concentrations and pool the fractions with the majority of the IgG. This can be done either spectrophotometrically or colorimetrically.
  5. Carry out the conjugation as soon as possible after this step (see Sample Experiment Protocol).
    Note     IgG solutions should be >4 mg/mL for the best results. The antibody should be concentrated if less than 2 mg/mL. Include an extra 10% for losses on the buffer exchange column.
    Note     The reduction can be carried out in almost any buffers from pH 7-7.5, e.g., MES, phosphate or TRIS buffers.
    Note     Steps 3 and 4 can be replaced by dialysis. 

SAMPLE EXPERIMENTAL PROTOCOL

This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with iFluor™ 647 maleimide. You might need further optimization for your particular proteins.
Note     Each protein requires distinct dye/protein ratio, which also depends on the properties of dyes. Over labeling of a protein could detrimentally affects its binding affinity while the protein conjugates of low dye/protein ratio gives reduced sensitivity.


Run conjugation reaction
  1. Use 10:1 molar ratio of Solution B (dye)/Solution A (protein) as the starting point:  Add 5 µL of the dye stock solution (Solution B, assuming the dye stock solution is 10 mM) into the vial of the protein solution (95 µL of Solution A) with effective shaking. The concentration of the protein is ~0.05 mM assuming the protein concentration is 10 mg/mL and the molecular weight of the protein is ~200KD.
    Note     We recommend to use 10:1 molar ratio of Solution B (dye)/Solution A (protein). If it is too less or too high, determine the optimal dye/protein ratio at 5:1, 15:1 and 20:1 respectively.
  2. Continue to rotate or shake the reaction mixture at room temperature for 30-60 minutes. 

Purify the conjugation
The following protocol is an example of dye-protein conjugate purification by using a Sephadex G-25 column.
  1. Prepare Sephadex G-25 column according to the manufacture instruction.
  2. Load the reaction mixture (From "Run conjugation reaction") to the top of the Sephadex G-25 column.
  3. Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
  4. Add more PBS (pH 7.2-7.4) to the desired sample to complete the column purification. Combine the fractions that contain the desired dye-protein conjugate.
    Note     For immediate use, the dye-protein conjugate need be diluted with staining buffer, and aliquoted for multiple uses.
    Note     For longer term storage, dye-protein conjugate solution need be concentrated or freeze dried. 

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of iFluor® 647 maleimide to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM82.553 µL412.763 µL825.525 µL4.128 mL8.255 mL
5 mM16.511 µL82.553 µL165.105 µL825.525 µL1.651 mL
10 mM8.255 µL41.276 µL82.553 µL412.763 µL825.525 µL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
/=x=

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Correction Factor (260 nm)0.03
Correction Factor (280 nm)0.03
Correction Factor (656 nm)0.0793
Extinction coefficient (cm -1 M -1)2500001
Excitation (nm)656
Emission (nm)670
Quantum yield0.251

Citations


View all 11 citations: Citation Explorer
Immune-regulating bimetallic metal-organic framework nanoparticles designed for cancer immunotherapy
Authors: Dai, Zan and Wang, Qiaoyun and Tang, Jie and Wu, Min and Li, Haoze and Yang, Yannan and Zhen, Xu and Yu, Chengzhong
Journal: Biomaterials (2022): 121261
Site-specific labeling and functional efficiencies of human fibroblast growth Factor-1 with a range of fluorescent Dyes in the flexible N-Terminal region and a rigid $\beta$-turn region
Authors: Mohale, Mamello and Gundampati, Ravi Kumar and Kumar, Thallapuranam Krishnaswamy Suresh and Heyes, Colin D
Journal: Analytical biochemistry (2022): 114524
SP/NK-1R Axis Promotes Perineural Invasion of Pancreatic Cancer and is Affected by lncRNA LOC389641
Authors: Ji, Tengfei and Ma, Keqiang and Wu, Hongsheng and Cao, Tiansheng
Journal: (2021)
Efferocytosis induces macrophage proliferation to help resolve tissue injury
Authors: Gerlach, Brennan D and Ampomah, Patrick B and Yurdagul Jr, Arif and Liu, Chuang and Lauring, Max C and Wang, Xiaobo and Kasikara, Canan and Kong, Na and Shi, Jinjun and Tao, Wei and others,
Journal: Cell metabolism (2021): 2445--2463
Enrichment of NPC1-deficient cells with the lipid LBPA stimulates autophagy, improves lysosomal function, and reduces cholesterol storage
Authors: Ilnytska, Olga and Lai, Kimberly and Gorshkov, Kirill and Schultz, Mark L and Tran, Bruce Nguyen and Jeziorek, Maciej and Kunkel, Thaddeus J and Azaria, Ruth D and McLoughlin, Hayley S and Waghalter, Miriam and others,
Journal: Journal of Biological Chemistry (2021)
Pharmacological targeting of Sam68 functions in colorectal cancer stem cells
Authors: Masibag, Angelique N and Bergin, Christopher J and Haebe, Joshua R and Zouggar, A{\"\i}cha and Shah, Muhammad S and Sandouka, Tamara and da Silva, Amanda Mendes and Desrochers, Fran{\c{c}}ois M and Fournier-Morin, Aube and Benoit, Yannick D
Journal: Iscience (2021): 103442
Enhanced Drug Delivery by Nanoscale Integration of a Nitric Oxide Donor to Induce Tumor Collagen Depletion
Authors: Dong, Xiao and Liu, Hai-Jun and Feng, Hai-Yi and Yang, Si-Cong and Liu, Xue-Liang and Lai, Xing and Lu, Qin and Lovell, Jonathan F and Chen, Hong-zhuan and Fang, Chao
Journal: Nano Letters (2019)
Deep Sequencing Analysis of the Eha-Regulated Transcriptome of Edwardsiella tarda Following Acidification
Authors: Gao, D and Liu, N and Li, Y and Zhang, Y and Liu, G and others, undefined
Journal: Metabolomics (Los Angel) (2017): 2153--0769
Influence of particle geometry on gastrointestinal transit and absorption following oral administration
Authors: Li, Dong and Zhuang, Jie and He, Haisheng and Jiang, Sifan and Banerjee, Amrita and Lu, Yi and Wu, Wei and Mitragotri, Samir and Gan, Li and Qi, Jianping
Journal: ACS applied materials \& interfaces (2017): 42492--42502
Suramin inhibits cullin-RING E3 ubiquitin ligases
Authors: Wu, Kenneth and Chong, Robert A and Yu, Qing and Bai, Jin and Spratt, Donald E and Ching, Kevin and Lee, Chan and Miao, Haibin and Tappin, Inger and Hurwitz, Jerard and others, undefined
Journal: Proceedings of the National Academy of Sciences (2016): E2011--E2018

References


View all 49 references: Citation Explorer
Sequential ordering among multicolor fluorophores for protein labeling facility via aggregation-elimination based beta-lactam probes
Authors: Sadhu KK, Mizukami S, Watanabe S, Kikuchi K.
Journal: Mol Biosyst (2011): 1766
Visualizing dengue virus through Alexa Fluor labeling
Authors: Zhang S, Tan HC, Ooi EE.
Journal: J Vis Exp. (2011)
Fluorescent "Turn-on" system utilizing a quencher-conjugated peptide for specific protein labeling of living cells
Authors: Arai S, Yoon SI, Murata A, Takabayashi M, Wu X, Lu Y, Takeoka S, Ozaki M.
Journal: Biochem Biophys Res Commun (2011): 211
Neuroanatomical basis of clinical joint application of "Jinggu" (BL 64, a source-acupoint) and "Dazhong" (KI 4, a Luo-acupoint) in the rat: a double-labeling study of cholera toxin subunit B conjugated with Alexa Fluor 488 and 594
Authors: Cui JJ, Zhu XL, Ji CF, Jing XH, Bai WZ.
Journal: Zhen Ci Yan Jiu (2011): 262
Simultaneous detection of virulence factors from a colony in diarrheagenic Escherichia coli by a multiplex PCR assay with Alexa Fluor-labeled primers
Authors: Kuwayama M, Shigemoto N, Oohara S, Tanizawa Y, Yamada H, Takeda Y, Matsuo T, Fukuda S.
Journal: J Microbiol Methods (2011): 119
Alexa Fluor 546-ArIB[V11L;V16A] is a potent ligand for selectively labeling alpha 7 nicotinic acetylcholine receptors
Authors: Hone AJ, Whiteaker P, Mohn JL, Jacob MH, McIntosh JM.
Journal: J Neurochem (2010): 994
Asymmetric trimethine 3H-indocyanine dyes: efficient synthesis and protein labeling
Authors: Song F, Wang L, Qiao X, Wang B, Sun S, Fan J, Zhang L, Peng X.
Journal: Org Biomol Chem (2010): 4249
Neuroanatomical characteristics of acupoint "Chengshan" (BL 57) in the rat: a cholera toxin subunit B conjugated with Alexa Fluor 488 method study
Authors: Zhu XL, Bai WZ, Wu FD, Jiang J, Jing XH.
Journal: Zhen Ci Yan Jiu (2010): 433
Photoactivatable and photoconvertible fluorescent probes for protein labeling
Authors: Maurel D, Banala S, Laroche T, Johnsson K.
Journal: ACS Chem Biol (2010): 507
Novel Alexa Fluor-488 labeled antagonist of the A(2A) adenosine receptor: Application to a fluorescence polarization-based receptor binding assay
Authors: Kecskes M, Kumar TS, Yoo L, Gao ZG, Jacobson KA.
Journal: Biochem Pharmacol (2010): 506